We have recently identified and begun to characterize a group of unique proteins, encoded by-the E3 region of human adenoviruses. These proteins, called 14.7K and 10.4K/14.5K, when expressed in virus- infected cells protect the cells from lysis by tumor necrosis factor (TNF). TNF is a multifunctional cytokine with pleiotropic effects ranging from cytolysis of transformed and infected cells in vitro to orchestration of the inflammatory response in vivo. Cytolysis by TNF occurs by apoptosis, resembling the programmed cell death seen during embryogenesis, and suggests that understanding of the intracellular signalling involved in the phenomenon may hold the key to a wide variety of important developmental signals. The mechanisms of TNF action have been extraordinarily difficult to dissect because of the diversity of effects it mediates. Thus, the identification of specific inhibitors that counteract some, but not all, TNF-induced responses introduces a new avenue of approach to studying the complex series of events that occurs following TNF binding to its cell surface receptor. We will continue our studies on the mechanism of action of these TNF inhibitory proteins. Specific aims are: (l) to evaluate the ability of the E3 proteins to function in transfected cells in the absence of other viral proteins and to produce a small panel of human and mouse cells lines expressing the E3 proteins for further study; (2) to elucidate structure/function relationships of the E3 proteins by analyzing a panel of 23 deletion and point mutants produced in the l0.4K gene. This aim will also determine the relationship between the two functions of 10.4K/14.5K, namely inhibition of TNF lysis and down- regulation of the EGF receptor; (3) to analyze the ability of the E3 proteins to inhibit selected functions associated with TNF cytolysis with emphasis on TNF receptor expression with I0.4KI14.5K and on modulation of cellular gene expression for 14.7K; (4) to use cDNA cloning to identify cellular proteins with which the E3 proteins interact in mediating TNF resistance. This aim takes advantage of our previous identification of variant cell lines in which the E3 proteins are expressed but do not prevent TNF lysis. We will introduce cDNA clones from normal cells by transfection into these variants expressing the E3 protein and use the powerful selection of TNF cytolysis to select resistant clones.